Which is best method for checking mycoplasma contamination in a mammalian cell line?
Mycoplasma contamination in mammalian cell cultures is difficult to detect by microscopy or culture because organisms lack a cell wall and grow slowly. PCR targeting conserved 16S rRNA or 16S-23S spacer regions offers highly sensitive, specific, and rapid detection of few genome copies in culture supernatant. Southern hybridization requires larger DNA amounts and is less sensitive. ELISA and Western hybridization detect proteins and lack broad Mycoplasma genus coverage. Hence PCR is the gold standard for routine screening mandated by cell banks, preventing altered growth, metabolism, and experimental artifacts.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.